Food and Bioproducts Processing 1 1 5 ( 2 0 1 9 ) 175–184

Contents lists available at ScienceDirect

Food and Bioproducts Processing

journal homepage: www.elsevier.com/locate/fbp

Solid-state fermentation as a sustainable method

for coffee pulp treatment and production of an
extract rich in chlorogenic acids

Jessica Santos da Silveira a,b , Noël Durand a,b , Stella Lacour c ,

Marie-Pierre Belleville c , Ana Perez d , Gérard Loiseau a , Manuel Dornier a,∗
a QualiSud, Univ Montpellier, CIRAD, Montpellier SupAgro, Univ Avignon, Univ Réunion, Montpellier, France
b CIRAD, UMR QualiSud, F-34398 Montpellier, France
c Institut Européen des Membranes, IEM – UMR 5635, CNRS, ENSCM, Univ Montpellier, Montpellier, France
d Universidad de Costa Rica, CITA, 11501-2060 San José, Costa Rica

a r t i c l e i n f o a b s t r a c t

Article history: In this study, coffee pulp was used as carbon source in solid-state fermentation to produce
Received 20 November 2018 a phenol-rich extract for industrial applications. Fermentations were carried out at labora-
Received in revised form 16 March tory (0.4 kg), semi-pilot (12 kg) and pilot (90 kg) scales in presence of 2.5 g kg−1 yeast strains
2019 and with three different coffee pulp materials. The extract stability was investigated using
Accepted 1 April 2019 different stabilizing agents: SO2 , ascorbic and acetic acids. Then, a study was conducted
Available online 9 April 2019 to determine the effect of ultrasound treatment on extraction yields. Results showed that
higher concentrations of chlorogenic acids were obtained with fermentation without ultra-
Keywords: sound treatment and by adding sulfite at 0.5 wt% at 8 h of fermentation. Despite of variations
Coffee pulp in coffee pulp composition, the process was validated at semi-pilot and pilot scales, pro-
Food waste treatment viding an extract 400% richer in chlorogenic acids (600 mg kg of coffee pulp) and with lower
Alcoholic fermentation sugar amounts to separate during the downstream processing.
Phenolic compounds extraction © 2019 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Chlorogenic acids

1. Introduction toxic and antinutritional to animals due to its polyphenols, tannins,

and caffeine content (Murthy and Madhava Naidu, 2012). Neverthe-
Worldwide, population growth has accelerated the agro-industrial less, coffee pulp is rich in sugars, proteins, minerals, amino-acids and
residues generation causing major environmental, economic and soci- also contains functional molecules of high industrial interest (pheno-
etal problems (water/soil/air pollution, toxicity, waste treatment and lic compounds with antioxidative, anti-inflammatory, antimutagenic,
disposal). The coffee production sector, one of the most important antibacterial, and anticancer properties; caffeine with well-known psy-
commodity in the world, generates huge amounts of residues, such chotropic and diuretic properties) (Shahidi and Chandrasekara, 2010;
as coffee pulp, husk, skin, among others (Esquivel and Jiménez, 2012), Arellano-González et al., 2011).
which contribute about 50% of the total coffee fruit production. Accord- Studies have shown that the data on coffee pulp composition can
ing to data from the International Coffee Organization (“ICO”, 2017), vary due to the characteristics of coffee fruits (cultivar, place of produc-
global green coffee production in 2017 was estimated to be around 9.5 tion, culture conditions, maturity, etc.), the postharvest management
million t with over 90% of coffee production taking place in developing (methods of depulping, drying, storage, etc.), or even the analytical
countries (Central and South America, Central and East Africa, Asia), methods used (Duangjai et al., 2016; Rios et al., 2014; Rodríguez-
whereas consumption is mainly in the industrialized economies. Cof- Durán et al., 2014). Four major classes of phenolic compounds were
fee pulp is the main residue (40% mass of coffee fruit) obtained from identified in recent studies: flavonols, flavan-3-ols, anthocyanidins,
the wet processing of coffee cherries and is considered as both eco- hydroxycinnamic acids and chlorogenic acids as the predominant
phenolic compound (Heeger et al., 2017). These molecules find evi-
dent applications in pharmaco-cosmetic, food and medical industries
∗
(Quideau et al., 2011). Moreover, hydroxycinnamic acids (HA) including
Corresponding author.
chlorogenic acids (CGA) can be converted into products of even higher
E-mail address: manuel.dornier@supagro.fr (M. Dornier).
https://doi.org/10.1016/j.fbp.2019.04.001
0960-3085/© 2019 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
176 Food and Bioproducts Processing 1 1 5 ( 2 0 1 9 ) 175–184

added value (vanillin hemisynthesis as an example) (Di Gioia et al., optimal catalytic activity. Moreover, it is well-known that hydroxycin-
2011). However, most of these molecules are found in coffee pulp con- namic acids are stable at lower pH (3–5), unlike basic conditions where
jugated to sugars as glycosides. Compounds such as chlorogenic acids isomerization and oxidation reactions can occur (pH ≥ 7.0) (Friedman
are found in an ester form bonded to cell wall forming highly complex and Jürgens, 2000; Ma et al., 2011; Narita and Inouye, 2013). A typical
polysaccharide structures (Asther et al., 2002). acidity regulator is acetic acid, an organic acid that has traditionally
Biotechnological processes such as solid-state fermentation can be been used to improve the shelf-life and microbiological safety of food
used to enhance the phenolic content in plant extracts through the products. Glacial acetic acid can potentially be used during fermenta-
breakage of ester bonds between phenolics and the plant cell wall, tion processes to enhance phenolic compounds extraction and stability
increasing their concentration and consequently functional properties by acidification of the medium, but can cause yeast death at certain
(Palmieri et al., 2018; Arellano-González et al., 2011). Solid-state fer- concentrations (Leão et al., 2001).
mentation is defined as any fermentation process performed on moist Based on these premises, the aim of this study was to investi-
solid materials in the absence of free-flowing water that acts both as gate the extraction and stabilization of chlorogenic acids from coffee
physical support and source of nutrients for microorganisms. Due to pulp during solid-state fermentation using commercial yeast strains.
this low water availability, a limited number of microorganisms, mainly First, the process optimization was investigated at laboratory scale
yeasts and fungi, can be used for solid-state fermentation (Thomas under well-controlled conditions. Then, the fermentation process was
et al., 2013). applied to larger scales in real condition of production in order to eval-
Saccharomyces cerevisiae is an important microorganism used world- uate its interest for industrial application. The outcomes of this study
wide for producing food and beverages. This yeast has several are expected to contribute to the development of a sustainable and
advantages for ethanol production from lignocellulosic biomass: effi- competitive method for coffee pulp treatment, applicable to all regions
cient ethanol production from simple sugars, it does not require where coffee is produced by the wet processing method (around 50%
oxygenation, has a relatively high tolerance to ethanol and inhibitors, of the worldwide production).
has low pH optimum and is generally recognized as safe (GRAS) as
a food additive for human consumption. The production of ethanol
during alcoholic fermentation also presents an advantage for the recov-
2. Materials and methods
ery of phenolic compounds from agri-food solid wastes, as mixtures
of water/ethanol have been shown to enhance the solubilization of 2.1. Raw material
phenols (Benmeziane et al., 2014).
Solid-state fermentation offers numerous advantages over other Coffee pulp from the wet depulping and demucilaging process
techniques such as lower energy requirements and the absence of of coffee beans (Coffea arabica) was supplied by the Ben-
sophisticated and complex machinery and control systems, providing eficio Coopeunión, a coffee-producer cooperative located in
a low-cost process in a sustainable framework. The main drawbacks Trés Rios (Costa Rica), during the 2015 and 2017 harvests
of this method concern the process scale-up (heat transfer and culture
for assays at laboratory and semi-pilot scale, respectively. As
homogeneity) and production yields. Several researchers have stud-
soon as obtained, raw coffee pulp was split into lots of 3 kg,
ied the application of ultrasound-assisted extraction to increase the
frozen at −20 ◦ C, and shipped to France by aircraft in a con-
conversion of starch materials to glucose as well as overall ethanol
yield during fermentation processes by destroying plant cell wall and trolled temperature system. Upon arrival, the bags containing
making it easier to microorganisms to access sugars from plant matrix frozen coffee pulp were stored at −20 ◦ C. Prior to experiments,
(Chemat et al., 2017; Nikolić et al., 2010). Ultrasonication has been batches of 1 kg of coffee pulp were thawed at room temper-
applied widely in various biological and chemical processes. However, ature and ground using a mixer Thermomix TM31 (Vorwerk,
the use of ultrasound-assisted extraction coupled to solid-state fer- Wuppertal, Germany) for 1 min at maximum power (level 10)
mentation to treat food waste has not been widely investigated. The use and without heating. The coffee pulp was then split into her-
of ultrasonics has the potential to break the pulp cell wall and to release metically sealed flasks and stored at −20 ◦ C.
sugars due to acoustic cavitation, enhancing ethanol production and
For the fermentation at pilot scale, coffee pulp was sup-
phenolic compounds recovery.
plied by the Beneficio San Diego, a coffee-producer cooperative
A major concern expressed with regards to the extraction of
located in San José (Costa Rica), during the 2018 harvest. The
polyphenols has been in relation to their stability. Phenolic compounds
may undergo degradation due to temperature, light, oxygen, enzymes,
night before the experiment, coffee pulp was collected and
and pH. Light and oxygen in the air are the two most important fac- stored at room temperature. Prior to the experiment, batches
tors that facilitate degradation reactions. Enzymes (mainly oxidative of 10 kg of coffee pulp were ground using a Bowl Cutter SM
enzymes) already present in the plant material can be released during 45 (K + G Wetter, Biedenkopf, Germany) for 7 min at maximum
the extraction process and promote such degradation reactions (Mäkilä power (level 2) and without heating. All batches of coffee pulp
et al., 2016). Therefore, to build an efficient extraction method, it is were gathered in a sterilized container and placed in a room
crucial to keep the stability of phenolic compounds. One way to avoid at 28 ◦ C.
phenol degradation during extraction is by adding a stabilizing agent
to the solution. In addition to presenting antifungal and antibacterial
properties, sulfur dioxide (SO2 ) is commonly used as a reducing agent 2.2. Chemicals
and as an inhibitor of endogenous oxidases in winemaking (Blouin,
2014; Ribereau-Gayon et al., 2006). Especially under its bisulfite form Methanol, sulfuric acid, and phosphoric acid were all of ana-
(HSO3 -), it can efficiently protect phenolics from chemical and enzy- lytical grade from Honeywell (Seelze, Germany). Standard
matic oxidations. Another well-known food preservative is ascorbic of chlorogenic acids containing a mix of 3-caffeoylquinic
acid, a water-soluble antioxidant naturally found in many fruits and acid (3-CQA), 5-caffeoylquinic acid (5-CQA), 4-caffeoylquinic
vegetables. In industry, ascorbic acid can be added to plant-based prod-
acid (4-CQA), 4-feruloylquinic acid (4-FQA), 5-feruloylquinic
ucts to preserve its antioxidant capacity and provide chemical stability.
acid (5-FQA), 3.4-dicaffeoylquinic acid (3.4-diCQA), 3.5-
Furthermore, in the presence of oxygen, ascorbic acid tends to oxidize,
dicaffeoylquinic acid (3.5-diCQA), and 4.5-dicaffeoylquinic
removing the environmental resources of oxygen (Varvara et al., 2016).
The acidification of the solution presents an alternative to prevent
acid (4.5-diCQA), was purchased from International Devel-
phenol degradation by adjusting the pH with acidity regulators. The opment and Manufacturing (New Jersey, USA). Standard of
pH optimum of enzymes, such as polyphenol oxidase (PPO), ranges glucose, fructose and ethanol were purchased from Sigma
around pH 6–7 and becomes inactive below pH 4. Hence, the role of Aldrich (Steinheim, Germany). l(+)-Ascorbic acid, glacial
acidity regulators is to maintain the pH well below that necessary for acetic acid, and sodium metabisulfite, used to stabilize
 Food and Bioproducts Processing 1 1 5 ( 2 0 1 9 ) 175–184 177

the chlorogenic acids during the assays, were all of ana- 2.5. Ultrasound-assisted extraction
lytical grade purchased from Sigma Aldrich (Steinheim,
Germany). For particular experiments, the fermentation at laboratory
scale was coupled to ultrasound-assisted extraction in order
to evaluate the possible enhancement of chlorogenic acid
2.3. Solid-state fermentation extraction. The ultrasound treatment was carried out using
a Bransonic model 3510E-MT 100 W ultrasound equipment
Two S. cerevisiae strains used for solid-state fermentation, with (Danbury, USA) using 42 kHz as frequency. Ultrasounds were
a population of 107 CFU g−1 , were kindly provided by Lalle- applied during 10 min (1) prior to inoculation or (2) at a selected
mand (Toulouse, France). The yeast strains were dried and time during fermentation where the maximum amount of
stored at 4 ◦ C. CGA was extracted. Total power used was 250 W/kg of coffee
Fermentations at laboratory scale were carried out using pulp.
a double-walled glass reactor (Legallais, Montferrier-sur-Lez,
France) with a working volume of 400 mL. The day prior to
2.6. Analytical methods
experiments, samples of 400 g of coffee pulp were thawed at
room temperature under darkness. The yeast strain was re-
The moisture of the raw coffee pulp was determined by gravi-
activated and multiplied using deionized water at 35 ◦ C for
metric method in triplicate at 100 ◦ C after 48 h. The pH and
30 min, at a ratio of 10 mL of water per g dried yeast. During
temperature were obtained using an ALMEMO measuring
this time, the coffee pulp was maintained at 28 ◦ C in the fer-
instrument (Ahlborn, Ilmenau, Germany). The concentration
mentation vessels. The temperature of the outer jacket was
of sugars and ethanol was determined by high-performance
controlled with a thermostatic water bath. Coffee pulp was
liquid chromatography (HPLC) using a Dionex Ultimate 3000
inoculated at a ratio of 2.5 g yeast/kg coffee pulp, and solid-
chromatograph (Thermo Scientific) that was equipped with a
state fermentation was carried out at a fixed temperature of
refractive index detector (RID-10A Schimadzu), a UV detector
28 ◦ C, in darkness and without stirring to maintain anaerobic
(210 nm), and an Aminex HPX87H column that was operated at
conditions during the fermentation. Prior to sampling, cof-
a temperature of 35 ◦ C using 5 mM sulfuric acid as the eluent
fee pulp was gently mixed using a sterilized laboratory tool
at a flow rate of de 0.6 mL min−1 .
to ensure homogeneity of the mixture. Samples were clarified
The concentration of chlorogenic acid isomers was deter-
by centrifugation at 8603 × g for 15 min.
mined by HPLC on a system consisting of Shimadzu (Kyoto,
Semi-pilot and pilot scale fermentations were carried
Japan) Model LC 20AD pumping units, an automated sam-
out respectively in France in a 20 L stainless steel tank
ple injector (Shimadzu SIL 20 AXR), a variable-wavelength UV
(Artame, Baguim do Monte, Portugal) containing 12 kg of cof-
detector (Shimadzu SPD20A), column Uptisphere type ODB 5
fee pulp and in Costa Rica in a 200 L sterilized polypropylene
(5 ␮m particle size, 250 × 4.6 mm), with identical pre-column,
drum (Lacoplast S.A., Guatemala) containing 90 kg of cof-
thermostatically controlled at 30 ◦ C. The elution program used
fee pulp. The tanks were placed in climate rooms set and
two solvents, A and B. Solvent A was 4 mM phosphoric acid and
preheated at 28 ◦ C for temperature control. Fermentations
solvent B was methanol. The following elution program was
were carried out at 28 ◦ C, without stirring and under dark-
used: A–B mixture 95/5 v/v from min 0 to 35, to A–B mixture
ness. Coffee pulp was inoculated, sampled and clarified as
25/75 v/v from min 35 to 40, then pure solvent B from min 40
described for laboratory scale. The coffee pulp extract was
to 50, to A–B mixture 95/5 v/v from min 50 to 55. Flow rate was
separated from the mash by pressing using a hydraulic press
1 mL min−1 . UV detection was at 327 nm, which corresponds
(Stossier LI P MO, Simaco, Bouzonville, France or TC Y125,
to maximum CGA absorption.
Owatonna Tool Company, Minnesota, USA) at 50–60 bar for
Samples were filtered through 0.45 ␮m pore size filter
30 min.
before HPLC injection. The compounds (sugars, ethanol, CGA)
were identified by comparison of their retention times with
2.4. Procedures for chlorogenic acid stabilization the retention times of certified standards. The quantification
of compounds was performed using calibration curves with 5
In order to prevent chlorogenic acid (CGA) degradation dur- different concentrations of standard solutions (R2 > 0.99).
ing the fermentation process, the performance of different
stabilizing agents was studied at laboratory scale. Sulfur diox- 3. Results and discussion
ide was used as sodium metabisulfite at 3 concentrations:
30 mg kg−1 according to data from the yeast supplier as the 3.1. Coffee pulp characteristics
limit sulfite concentration supported by their S. cerevisiae
strains, 500 mg kg−1 a concentration used for sulfitic macera- This study explored the fermentation of coffee pulp at three
tion of musts in winemaking that favors polyphenol extraction different scales: laboratory, semi-pilot, and pilot. Different
rate (Blouin, 2014), and 0.5 wt% a very high concentration batches of coffee pulp with different pretreatment condi-
which guarantees total protection against oxidation. Glacial tions were used during the experiments, which resulted in a
acetic acid was chosen as an acidity regulator at two concen- variability of the raw material composition. A chemical char-
trations: 1 wt% to stabilize CGA and allow yeast growth, and acterization of coffee pulp was carried out to determine its
at 10 wt% to stabilize CGA and inhibit yeast activity. Finally, initial composition in terms of fermentable sugars, ethanol
ascorbic acid at 1 wt% was used as an antioxidant (Narita and and chlorogenic acid 5-CQA (Table 1).
Inouye, 2013). The stabilizing agent was directly mixed to the The chlorogenic acid 5-CQA was reported as being the
coffee pulp before inoculation or at a certain time during the most abundant simple polyphenol present in fresh coffee
fermentation to study its effect on the stabilization of CGA and pulp (Martínez and Clifford, 2000) and its concentration was
on yeast activity. followed in all assays of this work. For laboratory scale experi-
178 Food and Bioproducts Processing 1 1 5 ( 2 0 1 9 ) 175–184

Table 1 – Characterization of raw coffee pulp from different batches in terms of origin, pretreatment steps, local and year
of assays, pH, moisture, sugar, ethanol and 5-CQA content.
Laboratory scale
Harvest year 2015 Supplier Coopeunion Freezing, grinding
Pretreatment steps
Year of assay(s) 2016–2017 Local of assay(s) France and freezing
Moisture [%] 85 ± 0.6
pH 4.4
Glucose [g L−1 ] Fructose [g L−1 ] Ethanol [g L−1 ] 5-CQA [mg L−1 ]
Upper value 35.16 48.35 0.85 12.7
Lower value 28.93 37.13 0.36 4.3
Average ()n 31.73 (1.84)8 42.97 (4.17)8 0.65 (0.15)8 7.93 (3.22)8

Semi-pilot scale
Harvest year 2017 Supplier Coopeunion Freezing, grinding
Pretreatment steps
Year of assay(s) 2018 Local of assay(s) France and freezing
Moisture [%] 85 ± 0.8
pH 4.3
Glucose [g L−1 ] Fructose [g L−1 ] Ethanol [g L−1 ] 5-CQA [mg L−1 ]
14.39 21.42 0.6 54.91

Pilot scale
Harvest year 2018 Supplier Beneficio San Diego
Pretreatment step Grinding
Year of assay(s) 2018 Local of assay(s) Costa Rica
Moisture [%] 85 ± 0.8
pH 4.3
Glucose [g L−1 ] Fructose [g L−1 ] Ethanol [g L−1 ] 5-CQA [mg L−1 ]
19.47 28.44 0.65 198.66

ments, eight samples of coffee pulp from the 2015 batch were
Table 2 – Relative concentration C/C0 (ratio between the
used. Fermentation at semi-pilot and pilot scales was car- final and initial concentrations) of glucose, fructose and
ried out only once due to limitations in coffee pulp supply. ethanol after 24 h of fermentation and final
According to results, we observed a difference in coffee pulp concentration of 5-CQA obtained at laboratory scale for
composition between samples of the same batch (2015) as well both yeast strains tested (Yeasts A and B) and for a
as significant differences between the three batches. Glucose control of coffee pulp non-inoculated.
and fructose contents varied by less than 10 % within the lab- Relative concentration Final concentration of
oratory scale samples whereas changed by 41% (glucose) and (C/C0 ) after 24 h 5-CQA after 24 h [mg L−1 ]
36% (fructose) between batches at different scales. Traces of Glucose Fructose Ethanol
ethanol were found in coffee pulp in concentrations inferior
Control 1.00 1.00 0.98 8
to 1 g L−1 , probably due to a natural (but very limited) fermen- Yeast A 0.01 0.11 42.80 30
tation of coffee pulp during storage. The biggest difference Yeast B 0.01 0.11 42.60 4
was found in terms of the amount of 5-CQA. We observed that
the highest concentration of 5-CQA, 198 mg L−1 , was found in
the 2018 batch that was only stored for one night prior to the chlorogenic acid extraction. Two strains were tested, Yeasts
experiment. Much lower concentrations were found in the cof- A and B, which differed in terms of sulfite production dur-
fee pulp batches of 2015 that were milled, frozen and stored ing fermentation (Yeast A presenting lower SO2 production).
for several months. An intermediate value was obtained for Table 2 presents the relative concentration (ratio between the
the batch of 2017 that was stored for a shorter period than the final and initial concentrations of a compound) for the glucose,
2015 batch. This can be explained by the high sensitivity of fructose, ethanol and 5-CQA after 24 h of fermentation.
phenolic compounds, such as chlorogenic acid 5-CQA, to iso- Results from the control (non-inoculated coffee pulp) show
merization and/or oxidation by external factors (temperature, that no alcoholic fermentation occurred within the 24 h period
light, oxygen) (Narita and Inouye, 2013; Xie et al., 2011) that and that the final coffee pulp extract contained 8.0 mg L−1 of
can occur during the pretreatment and storage steps, leading 5-CQA. Stability of the raw coffee pulp even with its endoge-
to CGA degradation. nous flora is probably correlated with its high phenolic content
This finding illustrates the importance of studying the ini- that inhibits microorganism growth. Both yeasts were able to
tial coffee pulp composition. Even though the same biomass ferment coffee pulp and lead to very similar performances
is used as a substrate for fermentation, quality of the raw in terms of sugar consumption and ethanol production. Nev-
material may lead to different ethanol production and chloro- ertheless, only one strain was capable of increasing the
genic acid extraction during fermentation, and this can be a concentration of 5-CQA in the extract from coffee pulp. While
challenge for industrial scale productions. the fermentation using Yeast A increased by almost 4 times
the concentration of 5-CQA compared to the control, results
3.2. Study of the fermentation process at with Yeast B showed the degradation of this molecule during
laboratory-scale the fermentation process. This result is probably correlated to
a difference in the production of pectinases, ␤-glucanases and
3.2.1. Selection of yeast strains and kinetics of CGA other hydrolytic enzymes that are able to facilitate the release
extraction of the 5-CQA out of plant cells (Alimardani-Theuil et al., 2011;
A preliminary study was carried out to select a S. cerevisiae Pinelo et al., 2006). This shows that only specific strains can
strain for coffee pulp fermentation with the view to enhance be used for the extraction of chlorogenic acids from coffee
 Food and Bioproducts Processing 1 1 5 ( 2 0 1 9 ) 175–184 179

inoculated with Yeast A and the results were compared to

a control experiment (non-inoculated coffee pulp). Fig. 1
presents the evolution of glucose, fructose and ethanol con-
centrations during the solid-state fermentation for both
samples.
Results showed that glucose was the preferred carbon
source, followed by fructose as usual. Both sugars were almost
completely consumed after 12 h of fermentation. The produc-
tion of ethanol was correlated to the consumption of sugars,
increasing during fermentation and levelling off to a final
concentration of 31.4 g L−1 (around 4% vol.) at t = 12 h, when
sugar stocks reached a very low level (glucose < 1 g L−1 ; fruc-
tose < 6 g L−1 ). No fermentation occurred in the control.
Fig. 2 presents the evolution of chlorogenic acids (CGA)
Fig. 1 – Substrate consumption (glucose, fructose) and extraction in the inoculated coffee pulp compared to the con-
ethanol production during solid-state fermentation of trol. The CGA identified were: 5-CQA, 4-CQA, 4-FQA, 5-FQA,
coffee pulp carried out at lab scale with Yeast A, compared and 4.5-diCQA. The evolution of the total amount of these five
to a control non-inoculated. chlorogenic acids is also presented (total CGA).
Results from the inoculated pulp (Yeast A) show that 5-CQA
was the only isomer that was significantly extracted during
pulp. The Yeast A was then selected for all the experiments the fermentation process. Its concentration increased over
presented in this work. time reaching a maximum at t = 8 h and decreasing afterward,
To better understand the fermentation and extraction pro- probably due to a higher degradation rate of this compound
cesses, a kinetic study was carried out with coffee pulp

Fig. 2 – Extraction of chlorogenic acids from coffee pulp during fermentation at lab scale with Yeast A compared to control
non-inoculated.
180 Food and Bioproducts Processing 1 1 5 ( 2 0 1 9 ) 175–184

there were either complete yeast inactivation resulting in no

chlorogenic acid extraction (500 mg kg−1 of SO2 ) or coffee pulp
fermentation but no chlorogenic acid stabilization (30 mg kg−1
of SO2 ). So, under those tested conditions, the stabilization
step applied to the coffee pulp before the fermentation process
was not suitable. A complementary study was then performed
comparing 3 different stabilizing agents (sulfite 0.5 wt%, ascor-
bic acid 1 wt%, and acetic acid 1 wt% and 10 wt%) added into
the culture medium at the optimal time of 5-CQA extraction,
i.e. at 8 h of fermentation (as previously shown in Fig. 2). This
new strategy aimed to stabilize the 5-CQA at its highest con-
centration in the extract and to study the effect of the addition
of stabilizing agent on the yeast activity.
Fig. 4 presents the evolution of glucose and 5-CQA concen-
Fig. 3 – Suggested competing pathways of the extraction trations for the different assays. Since 5 coffee pulp samples
and degradation of chlorogenic acids (CGA) during from batch 2015 were used during the experiments, the
fermentation of coffee pulp. concentrations were expressed in terms of the relative con-
centration of each compound (C/C0 ) due to raw material
compared to the extraction. The concentration of 4-CQA, 5- variability. Indeed, it is worth noting that, irrespective to the
FQA, and 4.5-diCQA varied very little over time, while the fermentation trials, fructose consumption and ethanol pro-
concentration of 4-FQA slowly decreased. The control showed duction showed the same evolution as previously described
similar behavior for the 4-CQA, 5-FQA, and 4.5-diCQA, with that confirmed the good reproducibility of the fermentation
their concentrations staying relatively stable over time. On the of the pulp by yeast. For this raison, the evolution of glucose
contrary the concentrations of 5-CQA and 4-FQA decreased concentration was used as the fermentation indicator in all
over time. During fermentation, different behaviors were the following results.
observed. Surprisingly for 5-CQA the concentration increased From Fig. 4, the addition of ascorbic acid at 1 wt% or acetic
over time reaching a maximum at 8 h and then decreased acid at 1 wt% didn’t affect yeast activity; the concentration of
drastically. It was already reported that the hydroxycinnamic glucose continued to decrease over time as it was observed
acids are mostly present in the coffee pulp under linked- in the experiment done without any stabilizing agent. As
form with cell walls whereas the soluble compounds could expected, the addition of sulfite at 0.5 wt% or acetic acid at
be mainly located inside the plant vacuoles (Rodríguez-Durán 10 wt% stopped the fermentation process, which indicates
et al., 2014). The enzymatic pool of the yeast which is respon- that, at the given concentration, sulfite and acetic acid inhib-
sible for the cell wall weakening could favor the wall-bonded ited yeast activity. Both of these stabilizing agents are known
5-CQA release as well as the increasing ethanol concentration for acting through a strong acidification of intracellular con-
could favor the solubility of this compound and its extraction. tent and, also by an intrinsic toxic activity like in the case of
Losses of 5-CQA become predominant after 8 h of fermen- HSO3 − , by blocking many carbonyl functions that disrupt the
tation through oxidation reactions by chemical, enzymatic cell metabolism (Blouin, 2014). Concerning the extraction of
and even fermentation pathways or through adsorption by 5-CQA, the addition of ascorbic acid tended to stabilize the 5-
the yeasts itselves (Mazauric and Salmon, 2005). Furthermore, CQA until 24 h of fermentation. Afterward, due to a gradually
with lower sugar stocks in the media, chlorogenic acids could oxidation of ascorbic acid, the 5-CQA started to degrade faster.
become the carbon source in its turn. Fig. 3 summarizes the The acetic acid, in both concentrations tested, was successful
possible pathways of extraction and degradation of CGA dur- to stabilize the 5-CQA. Surprisingly, the addition of sulfite not
ing coffee pulp fermentation. only stabilized the 5-CQA, but also increased its concentration
Finally, chlorogenic acids may also undergo various isomer- over time, reaching a maximum at 24 h of fermentation and
ization reactions (Liang and Kitts, 2015; Moon et al., 2009) and representing an enhancement of 400% of 5-CQA concentration
could explain the surprising stability observed in Fig. 2 for 4- compared to its initial concentration. This can be explained
CQA, 5-FQA and 4.5-diCQA, compared to 5-CQA and 4-CQA, first by the fact that 5-CQA can undergo reversible electro-
during fermentation (as an example, 5-CQA could be partially chemical reactions (Tomac and Šeruga, 2016), that is to say,
converted into 4-CQA and 5-FQA into 4-FQA). oxidized forms of 5-CQA can be reversibly reduced by the redox
To conclude this part of the study, these results showed that action of sulfite, unlike the other stabilizing agents tested in
it was possible to significantly extract the 5-CQA using solid- the study. Second, this result could also be due to a faster
state fermentation. In fact, alcoholic fermentation with Yeast destruction of the plant cells resulting in higher amounts
A allowed an increase of 3.5 times of 5-CQA initial concentra- of 5-CQA being released. This well-known phenomenon in
tion, from 4.3 mg L−1 to 15 mg L−1 , at only 8 h of fermentation. winemaking is often called the “dissolvent activity” of sulfites
However, in order to preserve the 5-CQA extracted during the (Ribereau-Gayon et al., 2006).
fermentation process, it is important to develop a stabilization Combining the results in terms of glucose consumption
method for chlorogenic acids. and 5-CQA extraction, two stabilizing agents could be used
for the fermentation of coffee pulp: acetic acid at 1 wt% that
3.2.2. Stabilization of chlorogenic acids allows yeast growth while stabilizing the 5-CQA gradually
A study was carried out to compare different strategies for the extracted, and the sulfite at 0.5 wt% that stops fermentation,
stabilization of chlorogenic acids in the coffee pulp. Initially, stabilizes and enhances 5-CQA extraction. For acetic acid, the
two concentrations of sulfite were chosen to stabilize coffee advantage is a complete consumption of sugars during the
pulp prior to inoculation: 30 and 500 mg kg−1 . It was found that fermentation. So, the low sugar content of the final extract
when adding this agent at the beginning of the fermentation, should considerably simplify the further purification steps
 Food and Bioproducts Processing 1 1 5 ( 2 0 1 9 ) 175–184 181

Fig. 4 – Effect of different stabilizing agents on the (A) glucose consumption and (B) 5-CQA extraction during solid-state
fermentation at lab scale with Yeast A (glucose: C0 = 32.5 ± 1.5 g L−1 ; 5-CQA: C0 = 5 ± 2 mg L−1 ).

during downstream processing. The use of sulfite will provide preserve the 5-CQA while allowing the fermentation process
an extract richer in chlorogenic acids but less pure (with more to continue.
sugars), and the use of acetic acid at 1 wt% will provide a purer Results showed that glucose consumption was similar for
extract, but with lower antioxidant capacity. Therefore, a com- both systems. Surprisingly, the glucose was not completely
promise must be found when choosing a stabilizing agent for consumed even after 48 h of fermentation, compared to previ-
this process. ous results that showed the glucose being mainly consumed
at 24 h of fermentation (Fig. 4). A moderate 5-CQA extrac-
tion optimization could be obtained with ultrasound-assisted
3.2.3. Study of the process optimization using
extraction, as the content of 5-CQA at 8 h of fermentation
ultrasound-assisted extraction
was only increased by around 280% (US at 8 h) or 220% (US
Two strategies were chosen to combine ultrasound-assisted
prior to fermentation) compared to 200% (fermentation with-
extraction to the fermentation process: prior to fermentation
out ultrasounds). Moreover, 5-CQA became unstable after
(i.e. before addition of yeasts) or after 8 h of fermentation when
24 h of fermentation, which could be explained by the pos-
the highest concentration of 5-CQA was achieved (according
sible release of endogenous oxidative enzymes due to the
to results in Section 3.2.1). Even if ultrasounds can disrupt also
ultrasound treatment. Another proposed explanation for the
the yeast cells causing yeast death and thus stop the fermen-
5-CQA instability relies on the fact that the ultrasound treat-
tation as it was recently reported (Chemat et al., 2017), the
ment could release other compounds that could interact with
fermentation during the first hours could potentiate ultra-
the 5-CQA throughout chemical reactions causing its degra-
sound effects and thus favor CGAs’ extraction. Since the
dation. Additionally, ultrasound treatment could enhance the
main purpose of our process was to enhance the amount of
extraction of many phenolic compounds from the coffee pulp
CGA in the coffee pulp extract, the use of ultrasounds dur-
that in turn could create an inhibitory environment for yeast
ing the fermentation was an interesting strategy to explore.
growth, leading to the decrease of fermentation activity. Under
Fig. 5 presents the results for glucose consumption and 5-CQA
the experimental conditions applied in this work, the coupling
extraction during the fermentation process. In that case, cof-
of ultrasounds prior or during the fermentation process is not
fee pulp was stabilized using acetic acid at 1 wt% in order to
182 Food and Bioproducts Processing 1 1 5 ( 2 0 1 9 ) 175–184

Fig. 5 – Effect of ultrasounds on glucose consumption (A) and 5-CQA extraction (B) during fermentation at laboratory-scale.
(Glucose: C0 = 31.9 ± 1.8 g L−1 ; 5-CQA: (C0 = 6 ± 2 mg L−1 ).

an interesting method to improve the extraction yields of 5-

CQA from coffee pulp. It didn’t improve the 5-CQA extraction
yield as expected, and even slowed down the fermentation
process. Therefore, no ultrasound treatment was applied on
further studies.

3.3. Application of the fermentation process at larger

scales

To evaluate the validity of the results obtained at laboratory

scale, fermentation at semi-pilot scale was carried out and
monitored during 24 h. Sulfite at 0.5 wt% was chosen as the
best stabilizing agent for the coffee pulp stabilization after a
first step of fermentation. As mentioned in Section 3.1, a dif- Fig. 6 – Glucose consumption and 5-CQA extraction during
ferent batch of coffee pulp was used for this assay (batch of fermentation at semi-pilot scale.
2017), which resulted in differences on the initial concentra-
tions of sugars and chlorogenic acids compared to the coffee of sugars in the coffee pulp from batch 2017 (14.39 g L−1 of glu-
pulp used for laboratory scale experiments (batch of 2015). The cose and 21.42 g L−1 of fructose) compared to the coffee pulp
batch of 2017, used for this study, contained lower concen- from batch 2015 (31.73 g L−1 of glucose and 42.97 g L−1 of fruc-
trations of sugars and a higher initial amount of 5-CQA, as tose). With most of the fermentable sugars being consumed
presented in the Table 1 and discussed in Section 3.1. Fig. 6 before 8 h of fermentation, the addition of sulfite at t = 8 h
presents the results for the glucose consumption and 5-CQA did not disturb the yeast activity since the fermentation was
extraction during the fermentation process. already completed at this time. The concentration of 5-CQA
The glucose was completely consumed after only 6 h of fer- increased over time as previously observed at laboratory scale,
mentation, which can be explained by the lower initial amount even after the addition of the sulfite (as explained in Section
 Food and Bioproducts Processing 1 1 5 ( 2 0 1 9 ) 175–184 183

very simple equipment can be used to obtain a valuable prod-

Table 3 – Overview of the year of coffee pulp batches, the
average temperature during the fermentation process (T) uct. It was important to develop a process that could be easily
and the relative concentration (ratio between the final C implemented in the coffee producing countries, without big
and initial C0 concentrations) of glucose, fructose, financial investment and operating costs, lower environmen-
ethanol and 5-CQA after 24 h of fermentation at tal impact and that could generate a new source of income for
laboratory, semi-pilot and pilot scales. the small coffee producers.
Scale Batch T [◦ C] Relative concentration (C/C0 ) at 24 h

Glucose Fructose Ethanol 5-CQA 4. Conclusions

Laboratory 2015 28 0.50 0.72 11.67 4.00 Solid-state fermentation was used to produce a valuable
Semi-pilot 2017 26 0.02 0.10 24.00 2.75
product from coffee pulp at laboratory, semi-pilot and pilot
Pilot 2018 22 0.14 0.22 22.86 3.09
scales. S. cerevisiae consumed the sugars releasing chlorogenic
acids that are found linked to the plant cell wall probably
3.3), up to a final concentration of 151 mg L−1 of 5-CQA at t as glycosides. Investigation of the extract stability and the
24 h, 275% higher than the initial concentration (55 mg L−1 of effect of ultrasounds revealed that higher extraction yields
5-CQA). When the fermentation was carried out at laboratory were obtained when fermentation was carried out without
scale, using sulfite at 0.5 wt% at t = 8 h as the stabilizing agent, ultrasound treatment and by using sulfite at 0.5 wt% as the sta-
the content of 5-CQA in the coffee pulp extract was increased bilizing agent. Irrespective of the scales, a phenol-rich extract
by 400% at t 24 h, against 275% at semi-pilot scale. Neverthe- was obtained containing 300–400% more chlorogenic acids
less, it was shown that the fermentation of coffee pulp and the than its initial concentration, within less than 24 h of fermen-
extraction of chlorogenic acids were successful at semi-pilot tation. After fermentation and solid/liquid separation steps,
scale, despite of the differences in coffee pulp composition the quantity of the remaining solid residue was considerably
(Table 1) and of the limitations encountered (reduced mass reduced. This final by-product will contain much less caffeine
and heat transfers during the process scale-up). and phenolics (compounds considered as anti-nutritional and
Finally, to evaluate the process in a coffee producing coun- eco-toxic), which could facilitate its reuse as animal feeding,
try, fermentation was carried out at pilot-scale in Costa Rica fertilizer or composting substrate.
with 90 kg of fresh coffee pulp nearby the green coffee produc- For further studies, it could be of great interest to test other
tion area. In order to compare the results from the semi-pilot yeast strains and to optimize fermentation conditions for even
fermentation, sulfite at a concentration of 0.5 wt% was used to increasing the extraction yield. In order to find the best sta-
stabilize the chlorogenic acids extracted during the fermen- bilizing method, in terms of (1) CGA preservation, (2) effect
tation. Table 3 summarizes the results for the fermentation on the fermentation activity and (3) cost and environmen-
performed at different scales. As mentioned in Section 3.1, the tal impact, new stabilizing agents must be tested at larger
coffee pulp composition varied due to the year of harvest and scales. To obtain a ready-to-market product, the concentra-
the storage time, so the relative concentration (C/C0 ) of each tion and the purification of the extract need to be performed
compound (glucose, fructose, ethanol and CGA) was chosen and investigated using sustainable technologies. Finally, the
instead of direct concentrations (C) values. characterization of the final product will have to be carried
For all three scales, sulfite at 0.5 wt% was used to stabilize out, e.g., in terms of antioxidant activity, to better determine
the 5-CQA extracted, added at 8 h of fermentation. At labora- its market added value and to enlighten about the economic
tory scale, the fermentation was immediately stopped when feasibility of the process.
sulfite was mixed to coffee pulp (Fig. 4), which explains the
higher relative concentrations of glucose and fructose in the Acknowledgements
product after 24 h. This was not the case for the 2017 and
2018 batches, which had lower initial concentrations of sug- This project was supported by Agropolis Fondation under the
ars, allowing the almost complete consumption of glucose and reference ID 1403-079 through the « Investissements d’avenir »
fructose prior to the addition of sulfite. In that cases, ethanol programme (Labex Agro: ANR-10-LABX-0001-01) » and Eurodia
production yields reached a quite high level between 0.37 and Industrie SA (Pertuis, France). The authors acknowledge the
0.41 g ethanol/g sugar which corresponds to 72 and 80% of the support from Fabrice Vaillant, researcher, and Joël Grabulos,
maximal theoretical conversion yield (0.51 g ethanol/g sugar). technician at CIRAD, for lending their facilities and for help
Nevertheless, the extraction of 5-CQA was very similar for all with the HPLC analyses. We also thank Lallemand (Toulouse,
three scales, with an augmentation of around 300% of its initial France) for providing S. cerevisiae strains.
concentration in less than 1 day of fermentation.
Although the operating temperature was set at 28 ◦ C, the
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